Total genomic DNA is extracted from normal human cells (generally skin fibroblast-derived cell lines, or circulating lymphocytes), tumors, or tumor-derived cell lines according to standard techniques. This DNA is screened for transforming activity by use of the NIH/3T3 transfection assay. Briefly, the DNA is coprecipitated with calcium phosphate and added to log phase cultures of NIH/3T3 cells. Transformation is screened morphologically 14-21 days after transfection, while foci which appear not be spontaneous are cloned with the glass cylinder method. DNA derived from these candidate foci is subjected to hybridization with a probe specific for human repetitive sequences. If no human repetitive sequences are found, the focus, the focus is felt to be spontaneous; if positive for human sequences, further hybridization with known transforming sequences is perormed. To date, this work has shown a minority of human tumors to contain activated transforming genes, detectable in this assay, in agreement with the experience of others: Kaposi's sarcoma, 0/5; chronic myelogenous leukemia, 0/15; diffuse histiocytic lymphoma, 0/6; hyperenphroma, 0/4; Hodgkin's lymphoma, 1/14; acute myelogenous leukemia. 1/1; rhabdomyosarcoma, 1/1. The latter two tumors were found to transform the murine target via transfer of the neuroblastoma-related onc gene n-ras (Shimizu et al., PNAS 80: 383, 1983). Another line of investigation has been to screen normal fibroblastic DNA from patients with high cancer risk syndromes (e.g., Gardner's syndrome to assess whether activation of cellular oncogenes occurs in normal tissues or is passed in germ line DNA. Such findings might account for familial cancer aggregation or multisite noplasia; however, to date we have found no evidence for active onc genes in normal tissues using this assay. An obvious limitation of these studies is the intrinsic limitation of the sensitivity of the assay, which seems skewed toward picking up genes of the ras family. Future studies are being directed toward modifying the assay to be more sensitivie, and thus hopefully broadening and increasing its ability to pick genes of the ras and other families.